The invention relates to an automated method and an apparatus for filling a piston cylinder with a variable but preset amount of sample solution and then discharging the sample solution from said piston cylinder onto a liquid chromatographic column.
Liquid chromatography is a well known separation technique which is utilized for separating various components contained in a sample. In the practice of liquid chromatography, it has been the custom to feed a measured amount of a sample solution containing a mixture of components needing to be separated to the top of a fractionation column of a chromatography material, e.g. gel particles, and then a suitable mobile phase (driving solution) is allowed to pass through the column, bringing with it components contained in the sample solution. The various components of the sample solution will, depending upon different degrees of interaction with the chromatography material, pass through the column at different speeds, thereby making it possible to separate the various components of the sample from each other.
A prerequisite for obtaining optimal results in liquid chromatographic separation is that the sample has to be applied on the column correctly. A common method of applying the sample solution onto the column is to apply the desired amount by means of an injection syringe. This manual method has obvious limitations.
Another prerequisite for obtaining optimal results in liquid chromatographic separation is that any appreciable intermixing of sample solution with driving solution prior to fractionation of the sample solution on the column should be avoided. If any appreciable intermixing of the sample solution with the driving solution occurs before actual fractionating of the sample solution on the column, such intermixing causes the separation bands to be fuzzy or to result in "tailing".
One method and apparatus for applying a variable but preset amount of sample solution to a chromatographic column is taught in U.S. Pat. No. 4,389,316 which makes use of a sample container of uniform cross-section, which by means of a piston is divided into a driving solution chamber and a sample solution chamber. The piston is slideable in the tubular container while sealing against the container wall so that the two chambers normally do not communicate with each other. Sample solution may be drawn into the sample solution chamber by moving the piston upwards in said sample container via a control handle attached to the piston rod which is attached to the piston. Said sample container may be marked with gradations so that the piston can be moved a preset amount in order to permit a fixed amount of sample solution to enter the sample solution chamber. Driving solution is then pumped into the driving solution chamber and this displaces the piston, thus discharging said fixed amount of sample solution to the chromatographic column. A few disadvantages to this method and apparatus are (1) difficulty of automation; (2) inability to vary the preset fixed amount of sample solution by small increments; (3) difficulty of providing a high pressure (e.g. 2000 psi) sliding seal to the outside, etc.
It is therefore an object of the invention to provide an automated method and apparatus for supplying a preset amount of sample solution to a liquid chromatographic column.
It is a further object of the invention to provide such a method and apparatus in which the preset amount of sample solution can be varied by small increments.
It is still a further object of the invention to provide such a method and apparatus in which any appreciable intermixing of sample solution with driving solution prior to fractionation of the sample solution on the column is avoided.
These and other advantages are obtained in accordance with the invention by means of the method and the apparatus for sample application defined in the following claims and described in more detail below.